The overall objective of this grant is to identify novel DNA methylation markers for detection of cervical intraepithelial neoplasia 3 (CIN-3) and carcinoma in situ (CIS), to access their clinical utility in identifying underlying CIN-3/CIS lesions among women with atypical squamous cells of undetermined significance (ASC-US) diagnosis, and to gain insight in the interaction between DNA methylation and specific histone modifications pre-marked by Polycomb group proteins. Despite the success of current cervical cancer control in the developed world, cytology screening lacks sensitivity and high-risk HPV (HR-HPV) testing specificity. Although the prophylactic HPV vaccine showed 100% efficacy for prevention of CIN-3/CIS due to HPV 16 and 18, it will not protect women from CIN-3/CIS caused by other high risk HPV types or protect women who are already infected with HPV 16 or 18. Therefore, screening will be necessary for the next several decades. However, current screening methods will be less cost-effective in the post-HPV vaccine era, when prevalence of CIN-3/CIS will decrease dramatically after elimination of HPV 16 and 18, which account for ~70% of ICC in the US and worldwide. We hypothesize that DNA methylation markers can be used to detect CIN-3/CIS with higher sensitivity and specificity than cytology and HR-HPV tests, and will have clinical utility in reducing colposcopy referral rate for women with a cytology diagnosis of ASC-US and a positive HR-HPV test. However, current available DNA methylation markers lack the sensitivity to detect CIN-3/CIS. Here we propose to use global methylation profiling to identify novel CIN-3/CIS-specific DNA methylation markers. First, primary short-term cultures will be established from CIN-3/CIS biopsy samples, epigenetically silenced genes will be reactivated using DNA methyltransferase and histone deacetylase inhibitors;and a set of reactivated genes, potentially hypermethylated specifically in CIN-3/CIS, will be identified through expression microarrays (Aim 1). The newly identified DNA methylation markers will be validated on archived normal, CIN- 1, CIN-2, CIN-3/CIS and ICC samples to derive a panel of markers for CIN-3/CIS/ICC detection (Aim 2), and their clinical utility in triage of women of ASC-US diagnosis will be determined (Aim 3). Based on our previous experience, we expect to identify six to ten novel CIN-3/CIS specific DNA methylation markers that will have sensitivity similar to HPV testing and specificity similar to cytology screening. These novel molecular biomarkers have the potential to improve the quality and cost-effectiveness of current cervical cancer screening strategies. The investigation of the role of Polycomb group proteins and chromatin modification in the origination of these DNA methylation changes will shed light on the hypothesis that cervical cancer derives from epithelial stem cells infected with HR-HPV and provide valuable information about cervical carcinogenesis. PUBLIC HEALTH RELEVANCE: Although the incidence of cervical cancer has been reduced by about 80% in the developed world through the implementation of cytology based screening to identify and treat cervical cancer precursor lesions, this approach is costly (estimated at a cost of $6 billion a year in the United States) and impractical in the developing world, where cervical cancer is still the leading cause of cancer death among women. We are proposing to identify novel DNA methylation markers that will be highly sensitive and specific to identify highly treatable cervical cancer precursor lesions. These markers will be able to reduce the over-treatment of women with mild abnormal cytological findings due to the low specificity of high-risk HPV testing. In the post-HPV vaccination era, these markers will be essential to provide a cost effective screening method for women already infected with HPV types that the current prophylactic vaccine does not provide protection against.